Project Summary/Abstract Plus end-directed kinesin and minus end-directed dynein move on microtubules in a stepwise manner using the energy from ATP hydrolysis and play crucial roles in axonal transport in neurons. They deliver a variety of cellular cargo such as vesicles, organelles, mRNA, and proteins along the microtubule tracks in the axons of neurons. Microtubule-based axonal transport is crucial, as the disruption of such intracellular transport causes neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, spastic paraplegia, and Huntington's disease. Both kinesin and dynein motors exist in an auto- inhibited conformation in the absence of cargo. Kinesin is auto-inhibited via a head-tail interaction that leads to extremely low microtubule binding frequency and slow and discontinuous motion on microtubules compared with a constitutively active kinesin. Dynein also exists in an auto-inhibited conformation in which the motor domains are closely stacked together and unable to move processively on microtubules. These molecular motors are attached to cargo via adapter proteins that specify a particular cargo. We recently showed that the adapter protein BicD binds dynein-dynactin and activates dynein for long distance transport only in the presence of both the mRNA binding protein Egalitarian and mRNA cargo. In Aim 1, we will similarly reconstitute a kinesin-BicD complex and investigate the mechanism by which BicD activates kinesin and regulates motor-driven cargo transport on microtubules. Using single- molecule approaches and TIRF microscopy, we will determine the domain on BicD that kinesin binds to, and the number of kinesins that can bind to BicD, which has a profound impact on its ability to perform long-distance transport. In cells, kinesin and dynein are both coupled via a common adapter protein and transport cargo through bidirectional motion on microtubules, by a mechanism that is not fully understood. In Aim 2, we will reconstitute kinesin-dynein complexes bound to a common BicD, label motors with different color Qdots, observe the motion, and monitor the stepping dynamics of each motor within the complex. We will investigate whether these two opposite polarity motors are engaged in a tug-of-war or if they coordinate during cargo transport, and whether motor number controls the directionality. These studies will provide a mechanistic framework for understanding axonal transport.